Vba2p, a vacuolar membrane protein involved in basic amino acid transport in Schizosaccharomyces pombe

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.     

Atg18 regulates organelle morphology and Fab1 kinase activity independent of its membrane recruitment by phosphatidylinositol 3,5-bisphosphate

The lipid kinase Fab1 governs yeast vacuole homeostasis by generating PtdIns(3,5)P(2) on the vacuolar membrane. Recruitment of effector proteins by the phospholipid ensures precise regulation of vacuole morphology and function. Cells lacking the effector Atg18p have enlarged vacuoles and high PtdIns(3,5)P(2) levels. Although Atg18 colocalizes with Fab1p, it likely does not directly interact with Fab1p, as deletion of either kinase activator-VAC7 or VAC14-is epistatic to atg18Delta: atg18Deltavac7Delta cells have no detectable PtdIns(3,5)P(2). Moreover, a 2xAtg18 (tandem fusion) construct localizes to the vacuole membrane in the absence of PtdIns(3,5)P(2), but requires Vac7p for recruitment. Like the endosomal PtdIns(3)P effector EEA1, Atg18 membrane binding may require a protein component. When the lipid requirement is bypassed by fusing Atg18 to ALP, a vacuolar transmembrane protein, vac14Delta vacuoles regain normal morphology. Rescue is independent of PtdIns(3,5)P(2), as mutation of the phospholipid-binding site in Atg18 does not prevent vacuole fission and properly regulates Fab1p activity. Finally, the vacuole-specific type-V myosin adapter Vac17p interacts with Atg18p, perhaps mediating cytoskeletal attachment during retrograde transport. Atg18p is likely a PtdIns(3,5)P(2)"sensor," acting as an effector to remodel membranes as well as regulating its synthesis via feedback that might involve Vac7p.     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.     

Atg27 Tyrosine Sorting Motif is Important for Its Trafficking and Atg9 Localization

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9‐positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP‐3, which mediates direct transport from the trans‐Golgi to the vacuole. The four C‐terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP‐3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.      

Atg19 mediates a dual interaction cargo sorting mechanism in selective autophagy

Autophagy is a catabolic membrane-trafficking mechanism conserved in all eukaryotic cells. In addition to the nonselective transport of bulk cytosol, autophagy is responsible for efficient delivery of the vacuolar enzyme Ape1 precursor (prApe1) in the budding yeast Saccharomyces cerevisiae, suggesting the presence of a prApe1 sorting machinery. Sequential interactions between Atg19-Atg11 and Atg19-Atg8 pairs are thought responsible for targeting prApe1 to the vesicle formation site, the preautophagosomal structure (PAS), and loading it into transport vesicles, respectively. However, the different patterns of prApe1 transport defect seen in the atg11Delta and atg19Delta strains seem to be incompatible with this model. Here we report that prApe1 could not be targeted to the PAS and failed to be delivered into the vacuole in atg8Delta atg11Delta double knockout cells regardless of the nutrient conditions. We postulate that Atg19 mediates a dual interaction prApe1-sorting mechanism through independent, instead of sequential, interactions with Atg11 and Atg8. In addition, to efficiently deliver prApe1 to the vacuole, a proper interaction between Atg11 and Atg9 is indispensable. We speculate that Atg11 may elicit a cargo-loading signal and induce Atg9 shuttling to a specific PAS site, where Atg9 relays the signal and recruits other Atg proteins to induce vesicle formation.     

Vba2p,A Vacuolar Membrane Protein Involved in Basic Amino Acid Transport in Schizosaccharomyces pombe

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.     

Permeases recycle amino acids resulting from autophagy

Autophagy is a major survival mechanism for eukaryotes to recycle cellular nutrients during stress conditions (such as nutrient limitation, or the accumulation of damaged organelles). We recently revealed a molecular mechanism by which Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Atg22 is not directly required for autophagic body breakdown, in contrast to previously reported data. Instead, we found that Atg22, Avt3 and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagy.     

Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway

Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.     

Vesicle-mediated protein transport pathways to the vacuole in Schizosaccharomyces pombe

The vacuole of Saccharomyces cerevisiae plays essential roles not only for osmoregulation and ion homeostasis but also down-regulation (degradation) of cell surface proteins and protein and organellar turnover. Genetic selections and genome-wide screens in S. cerevisiae have resulted in the identification of a large number of genes required for delivery of proteins to the vacuole. Although the complete genome sequence of the fission yeast Schizosaccharomyces pombe has been reported, there have been few reports on the proteins required for vacuolar protein transport and vacuolar biogenesis in S. pombe. Recent progress in the S. pombe genome project of has revealed that most of the genes required for vacuolar biogenesis and protein transport are conserved between S. pombe and S. cerevisiae. This suggests that the basic machinery of vesicle-mediated protein delivery to the vacuole is conserved between the two yeasts. Identification and characterization of the fission yeast counterparts of the budding yeast Vps and Vps-related proteins have facilitated our understanding of protein transport pathways to the vacuole in S. pombe. This review focuses on the recent advances in vesicle-mediated protein transport to the vacuole in S. pombe.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.     

Permeases Recycle Amino Acids Resulting from Autophagy

Autophagy is a major survival mechanism for eukaryotes to recycle cellular nutrients during stress conditions (such as nutrient limitation, or the accumulation of damaged organelles). We recently revealed a molecular mechanism by which Atg22 recycles amino acids to link the degradative and recycling functions of autophagy. Atg22 is not directly required for autophagic body breakdown, in contrast to previously reported data. Instead, we found that Atg22, Avt3 and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagy.

Addendum to:

Atg22 Recycles Amino Acids to Link the Degradative and Recycling Functions of Autophagy

Zhifen Yang, Ju Huang, Jiefei Geng, Usha Nair and Daniel J. Klionsky

Mol Biol Cell 2006; Oct. 4     

Fission yeast syt22 protein, a putative Arf guanine nucleotide exchange factor, is necessary for new end take off

In fission yeast Schizosaccharomyces pombe , the directions of cell growth change from monopolar to bipolar in character, which is known as 'new end take off' (NETO). We previously found that arf6p, a member (class III) of the ADP-ribosylation factor (Arf) family, is necessary for NETO in fission yeast. Here we report the characterization of an S. pombe gene, syt22 ⁺, encoding a putative Arf guanine nucleotide exchange factor (GEF). The syt22 protein contains a Sec7 domain and a PH domain conserved in the mammalian EFA6 GEF family, and has high similarity to Yel1p, which was identified as a GEF for Arf3p (class III Arf) in Saccharomyces cerevisiae. syt22 Δ cells, like arf6 Δ cells, completely failed to undergo NETO. Syt22p uniformly localizes to the cell periphery. Its localization is not dependent on microtubules, actin cytoskeletons or arf6p. We hypothesize that syt22p functions as a GEF for arf6p.     

The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 of Saccharomyces cerevisiae.

The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and mating-type switching. We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect. An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses. The rad22 gene contains no intron. A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae. A rad22 disruption strain was constructed which seems to be inviable in a homothallic background. Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region. These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks.     

ATG23, a novel gene required for maturation of proaminopeptidase I, but not for autophagy

In rich media proaminopeptidase I is targeted to the vacuole via the Cvt pathway and during starvation via autophagy. We here identify Atg23 (Ylr431c), a protein of so far unknown function, as a novel component essential for proaminopeptidase I maturation under non-starvation conditions. Maturation of proaminopeptidase I takes place in starved atg23Delta cells. Selective vacuolar targeting of the autophagosomal marker GFP-Aut7 and the accumulation of autophagic bodies during starvation in the presence of phenylmethylsulfonyl fluoride suggest that autophagy occurs in atg23Delta cells but at a reduced rate. In atg23Delta cells mature vacuolar carboxypeptidase Y is present and accumulation of quinacrine suggests no significant defect in vacuolar acidification. Furthermore, growth of atg23Delta cells on nitrocellulose detects no significant secretion of carboxypeptidase Y.     

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.     

An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination

Yeast (Saccharomyces cerevisiae) Atg6/Vps30 is required for autophagy and the sorting of vacuolar hydrolases, such as carboxypeptidase Y. In higher eukaryotes, however, roles for ATG6/VPS30 homologs in vesicle sorting have remained obscure. Here, we show that AtATG6, an Arabidopsis (Arabidopsis thaliana) homolog of yeast ATG6/VPS30, restored both autophagy and vacuolar sorting of carboxypeptidase Y in a yeast atg6/vps30 mutant. In Arabidopsis cells, green fluorescent protein-AtAtg6 protein localized to punctate structures and colocalized with AtAtg8, a marker protein of the preautophagosomal structure. Disruption of AtATG6 by T-DNA insertion resulted in male sterility that was confirmed by reciprocal crossing experiments. Microscopic analyses of AtATG6 heterozygous plants (AtATG6/atatg6) crossed with the quartet mutant revealed that AtATG6-deficient pollen developed normally, but did not germinate. Because other atatg mutants are fertile, AtAtg6 likely mediates pollen germination in a manner independent of autophagy. We propose that Arabidopsis Atg6/Vps30 functions not only in autophagy, but also plays a pivotal role in pollen germination.     

pdf1, a palmitoyl protein thioesterase 1 Ortholog in Schizosaccharomyces pombe: a yeast model of infantile Batten disease

Infantile Batten disease is a severe neurodegenerative storage disorder caused by mutations in the human PPT1 (palmitoyl protein thioesterase 1) gene, which encodes a lysosomal hydrolase that removes fatty acids from lipid-modified proteins. PPT1 has orthologs in many species, including lower organisms and plants, but not in Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe contains a previously uncharacterized open reading frame (SPBC530.12c) that encodes the S. pombe Ppt1p ortholog fused in frame to a second enzyme that is highly similar to a previously cloned mouse dolichol pyrophosphatase (Dolpp1p). In the present study, we characterized this interesting gene (designated here as pdf1, for palmitoyl protein thioesterase-dolichol pyrophosphate phosphatase fusion 1) through deletion of the open reading frame and complementation by plasmids bearing mutations in various regions of the pdf1 sequence. Strains bearing a deletion of the entire pdf1 open reading frame are nonviable and are rescued by a pdf1 expression plasmid. Inactivating mutations in the Dolpp1p domain do not rescue the lethality, whereas mutations in the Ppt1p domain result in cells that are viable but abnormally sensitive to sodium orthovanadate and elevated extracellular pH. The latter phenotypes have been previously associated with class C and class D vacuolar protein sorting (vps) mutants and vacuolar membrane H(+)-ATPase (vma) mutants in S. cerevisiae. Importantly, the Ppt1p-deficient phenotype is complemented by the human PPT1 gene. These results indicate that the function of PPT1 has been widely conserved throughout evolution and that S. pombe may serve as a genetically tractable model for the study of human infantile Batten disease.     

Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe

Recombinant plasmids were constructed that direct the synthesis of human antithrombin III in baker's yeast, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. The signal sequence of antithrombin III was recognized by both yeast species, and antithrombin III was secreted into the medium. When the signal sequence was replaced by a sequence of ten arbitrary amino acids, the product expressed from such a construct stayed inside the cell. Antithrombin III was glycosylated by the baker's and fission yeast and was immunologically identical to antithrombin III isolated from human plasma. Antithrombin III isolated from the culture media of recombinant yeasts was biologically active, as could be shown by progressive inhibitor activity and heparin cofactor activity.     

Assortment of phosphatidylinositol 3-kinase complexes--Atg14p directs association of complex I to the pre-autophagosomal structure in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.